Development and validation of prognostic machine learning models for short- and long-term mortality among acutely admitted patients based on blood tests.

Several scores predicting mortality at the emergency department have been developed. However, all with shortcomings either simple and applicable in a clinical setting, with poor performance, or advanced, with high performance, but clinically difficult to implement. This study aimed to explore if machine learning algorithms could predict all-cause short- and long-term mortality based on the routine blood test collected at admission. METHODS: We analyzed data from a retrospective cohort study, including patients > 18 years admitted to the Emergency Department (ED) of Copenhagen University Hospital Hvidovre, Denmark between November 2013 and March 2017. The primary outcomes were 3-, 10-, 30-, and 365-day mortality after admission. PyCaret, an automated machine learning library, was used to evaluate the predictive performance of fifteen machine learning algorithms using the area under the receiver operating characteristic curve (AUC). RESULTS: Data from 48,841 admissions were analyzed, of these 34,190 (70%) were randomly divided into training data, and 14,651 (30%) were in test data. Eight machine learning algorithms achieved very good to excellent results of AUC on test data in a of range 0.85-0.93. In prediction of short-term mortality, lactate dehydrogenase (LDH), leukocyte counts and differentials, Blood urea nitrogen (BUN) and mean corpuscular hemoglobin concentration (MCHC) were the best predictors, whereas prediction of long-term mortality was favored by age, LDH, soluble urokinase plasminogen activator receptor (suPAR), albumin, and blood urea nitrogen (BUN). CONCLUSION: The findings suggest that measures of biomarkers taken from one blood sample during admission to the ED can identify patients at high risk of short-and long-term mortality following emergency admissions.

Biomarkers and the post-thrombotic syndrome: A systematic review of biomarkers associated with the occurrence of the post-thrombotic syndrome after lower extremity deep venous thrombosis.

INTRODUCTION: Post-thrombotic syndrome (PTS) is a frequent chronic complication of deep venous thrombosis (DVT). Biomarkers are potentially valuable clinical tools for handling PTS. The purpose of this review was to examine which biomarkers are associated with the development of PTS in adults with lower extremity DVT. METHODS: We performed a systematic review of all English language prospective studies of biomarkers and PTS published in PubMed and EMBASE. Studies were included if diagnosing DVT by diagnostic imaging and assessing PTS by clinical scales, for example, the Villalta scale. Biomarkers of thrombophilia and pathological clot properties were not assessed. Data was reported qualitatively. RESULTS: 15 prospective studies were included. Studies varied widely in study design and methods of data analysis. Forty-six different biomarkers were examined, with seven being measured in two or more studies. The most frequently studied biomarkers were D-dimer, CRP, and IL-6. Associations between PTS and D-dimer were predominantly significant, while results on CRP and IL-6 were inconsistent. ICAM-1 was consistently associated with PTS in all studies and at all timepoints. IL-10 was significantly related to PTS development in the largest study and at all time points. Adiponectin, tPA, HRG and TAFI, MMP-1 and -8, and TIMP-1 and -2 were significantly associated with PTS in single studies. CONCLUSION: (1) Further research on biomarkers and PTS is clearly warranted. (2) Significant differences in study designs made it difficult to draw reliable conclusions regarding individual biomarkers. We suggest the implementation of a standardized framework for the study of biomarkers and PTS, to make comparison of future studies more feasible. (3) D-dimer, ICAM-1, IL-10, MMP-1 and 8, TIMP-1, TIMP-2, and adiponectin are clinical biomarkers of particular interest to include in future studies of PTS. Large scale systemic quantitative proteomic analyses of DVT patients could help identify novel biomarkers of interest in PTS-patients.

Senolytic and senomorphic interventions to defy senescence-associated mitochondrial dysfunction.

The accumulation of senescent cells in the aging individual is associated with an increase in the occurrence of age-associated pathologies that contribute to poor health, frailty, and mortality. The number and type of senescent cells is viewed as a contributor to the body's senescence burden. Cellular models of senescence are based on induction of senescence in cultured cells in the laboratory. One type of senescence is triggered by mitochondrial dysfunction. There are several indications that mitochondria defects contribute to body aging. Senotherapeutics, targeting senescent cells, have been shown to induce their lysis by means of senolytics, or repress expression of their secretome, by means of senomorphics, senostatics or gerosuppressors. An outline of the mechanism of action of various senotherapeutics targeting mitochondria and senescence-associated mitochondria dysfunction will be here addressed. The combination of geroprotective interventions together with senotherapeutics will help to strengthen mitochondrial energy metabolism, biogenesis and turnover, and lengthen the mitochondria healthspan, minimizing one of several molecular pathways contributing to the aging phenotype.

Performance of Panel-Estimated GFR Among Hospitalized Older Adults.

RATIONALE & OBJECTIVE: Older adults represent nearly half of all hospitalized patients and are vulnerable to inappropriate dosing of medications eliminated through the kidneys. However, few studies in this population have evaluated the performance of equations for estimating the glomerular filtration rate (GFR)-particularly those that incorporate multiple filtration markers. STUDY DESIGN: Cross-sectional diagnostic test substudy of a randomized clinical trial. SETTING & PARTICIPANTS: Adults≥65 years of age presenting to the emergency department of Copenhagen University Hospital Amager and Hvidovre in Hvidovre, Denmark, between October 2018 and April 2021. TESTS COMPARED: Measured GFR (mGFR) determined using 99mTc-DTPA plasma clearance compared with estimated GFR (eGFR) calculated using 6 different equations based on creatinine; 3 based on creatinine and cystatin C combined; and 2 based on panels of markers including creatinine, cystatin C, ß-trace protein (BTP) and/or ß2-microglobulin (B2M). OUTCOME: The performance of each eGFR equation compared with mGFR with respect to bias, relative bias, inaccuracy (1-P30), and root mean squared error (RMSE). RESULTS: We assessed eGFR performance for 106 patients (58% female, median age 78.3 years, median mGFR 62.9mL/min/1.73m2). Among the creatinine-based equations, the 2009 CKD-EPIcr equation yielded the smallest relative bias (+4.2%). Among the creatinine-cystatin C combination equations, the 2021 CKD-EPIcomb equation yielded the smallest relative bias (-3.4%), inaccuracy (3.8%), and RMSE (0.139). Compared with the 2021 CKD-EPIcomb, the CKD-EPIpanel equation yielded a smaller RMSE (0.136) but larger relative bias (-4.0%) and inaccuracy (5.7%). LIMITATIONS: Only White patients were included; only a subset of patients from the original clinical trial underwent GFR measurement; and filtration marker concentration can be affected by subclinical changes in volume status. CONCLUSIONS: The 2009 CKD-EPIcr, 2021 CKD-EPIcomb, and CKD-EPIpanel equations performed best and notably outperformed their respective full-age spectrum equations. The addition of cystatin C to creatinine-based equations improved performance, while the addition of BTP and/or B2M yielded minimal improvement. FUNDING: Grants from public sector industry (Amgros I/S) and government (Capital Region of Denmark). TRIAL REGISTRATION: Registered at ClinicalTrials.gov with registration number NCT03741283. PLAIN-LANGUAGE SUMMARY: Inaccurate kidney function assessment can lead to medication errors, a common cause of hospitalization and early readmission among older adults. Several novel methods have been developed to estimate kidney function based on a panel of kidney function markers that can be measured from a single blood sample. We evaluated the accuracy of these new methods (relative to a gold standard method) among 106 hospitalized older adults. We found that kidney function estimates combining 2 markers (creatinine and cystatin C) were highly accurate and noticeably more accurate than estimates based on creatinine alone. Estimates incorporating additional markers such as ß-trace protein and ß2-microglobulin did not further improve accuracy.

Successful isolation of viable stem cells from cryopreserved microfragmented human adipose tissue from patients with knee osteoarthritis - a comparative study of isolation by tissue explant culture and enzymatic digestion.

PURPOSE: To investigate if viable stem cells could be isolated and expanded from cryopreserved microfragmented adipose tissue (AT) harvested from patients with knee osteoarthritis. METHODS: Microfragmented abdominal AT from knee osteoarthritis patients was cryopreserved at -80 °C in cryoprotectant-medium. The samples were thawed for stem cell isolation by tissue explant culture (TEC) and enzymatic digestion (ED), respectively. Viability, population doublings, and doubling time were assessed by trypan blue staining and flow cytometry. Cell type and senescence-associated ß-galactosidase activity were analyzed by flow cytometry. Osteogenic and adipogenic differentiation was assessed quantitatively by Alizarin-Red-S and Oil-Red-O staining, respectively. RESULTS: Microfragmented AT from 7 patients was cryopreserved for a period of 46-150 days (mean (SD) 115.9 days (44.3 days)). Viable stem cells were successfully recovered and expanded from all patients using both isolation methods with no significant difference in viable population doublings or doubling time from passage 1 to 3 (p > 0.05). Low levels of senescence-associated ß-galactosidase activity was detected for both methods with no significant difference between TEC and ED (p = 0.17). Stemness was verified by stem cell surface markers and osteogenic and adipogenic differentiation performance. Adventitial stem cells (CD31-CD34+CD45-CD90+CD146-), pericytes (CD31-CD34-CD45-CD90+CD146+), transitional pericytes (CD31-CD34+CD45-CD90+CD146+), and CD271+ stem cells (CD31-CD45-CD90+CD271+) were identified using both methods. More pericytes were present when using TEC (25% (24%)) compared to ED (3% (2%)) at passage 4 (p = 0.04). CONCLUSIONS: Viable stem cells can be isolated and expanded from cryopreserved microfragmented AT using both TEC and ED. TEC provides more clinically relevant pericytes than ED.

Soluble urokinase plasminogen activator receptor and decline in kidney function among patients without kidney disease.

Background: Hospitalized patients are at an increased risk of developing kidney disease after discharge, often despite the absence of any clinical indicators during hospitalization. Soluble urokinase plasminogen activator receptor (suPAR) is a marker of systemic chronic inflammation that can be measured from routine blood samples. We determined whether elevated suPAR during hospitalization is associated with a decline in estimated glomerular filtration rate (eGFR) after discharge. Methods: This was a retrospective longitudinal cohort study of patients without detectable kidney disease presenting to the emergency department on two separate occasions during a 3-year period. The association between suPAR and a decline in eGFR was assessed by linear mixed models for repeated measures adjusting for age, sex, C-reactive protein, sodium, diabetes, hypertension and cardiovascular disease. Results: In total, 5124 patients (median age 65.9 years, 51.0% female) were included. The median suPAR was 2.9 ng/mL, the median time to readmission was 144 days and the expected rate of eGFR decline over this period was 5.1 mL/min/1.73 m2/year. Adjusting for other risk factors, patients with suPAR <3, 3-6 or ≥6 ng/mL had an expected eGFR decline of 4.3, 5.2 or 9.0 mL/min/1.73 m2/year, respectively. Similarly, patients with suPAR in the lowest (<2.4 ng/mL), middle (2.4-3.6 ng/mL) or highest (≥3.6 ng/mL) tertile had an expected eGFR decline of 4.2, 4.6 or 6.5 mL/min/1.73 m2/year, respectively. In both cases, a higher suPAR level was significantly and independently associated with a higher rate of eGFR decline (P < .001). Conclusions: A higher suPAR level was associated with accelerated eGFR decline among patients presenting to the emergency department, suggesting that routine suPAR measurements may have utility for the early detection of kidney disease.

suPAR Cut-Offs for Risk Stratification in Patients With Symptoms of COVID-19.

OBJECTIVES: Elevated soluble urokinase Plasminogen Activator Receptor (suPAR) is a biomarker associated with adverse outcomes. We aimed to investigate the associations between plasma suPAR levels (testing the cut-offs ⩽4, 4-6, and ⩾6 ng/mL) with risk of 14-day mortality, and with the risk of mechanical ventilation in patients that tested positive for SARS-CoV-2. METHODS: Observational cohort study of patients presenting with symptoms of COVID-19 at Department of Emergency Medicine, Amager and Hvidovre Hospital, Denmark from March 19th, 2020 to April 3rd, 2020. Plasma suPAR was measured using suPARnostic technologies. Patients were followed for development of mechanical ventilation and mortality for 14 days. Validation of our findings were carried out in a similar sized COVID-19 patient cohort from Mikkeli Central Hospital, Finland. RESULTS: Among 386 patients with symptoms of COVID-19, the median (interquartile range) age was 64 years (46-77), 57% were women, median suPAR was 4.0 ng/mL (2.7-5.9). In total, 35 patients (9.1%) died during the 14 days follow-up. Patients with suPAR ⩽4 ng/mL (N = 196; 50.8%) had a low risk of mortality (N = 2; 1.0%; negative predictive value of 99.0%, specificity 55.3%, sensitivity 95.2%, positive predictive value 17.4%). Among patients with suPAR ⩾6 ng/mL (N = 92; 23.8%), 16 died (17.4%). About 99 patients (25.6%) tested positive for SARS CoV-2 and of those 12 (12.1%) developed need for mechanical ventilation. None of the SARS-CoV-2 positive patients with suPAR ⩽4 ng/mL (N = 28; 38.8%) needed mechanical ventilation or died. The Mikkeli Central Hospital validation cohort confirmed our findings concerning suPAR cut-offs for risk of development of mechanical ventilation and mortality. CONCLUSIONS: Patients with symptoms of COVID-19 and suPAR ⩽4 or ⩾6 ng/mL had low or high risk, respectively, concerning the need for mechanical ventilation or mortality. We suggest cut-offs for identification of risk groups in patients presenting to the ED with symptoms of or confirmed COVID-19.

Soluble Urokinase Plasminogen Activator Receptor as a Decision Marker for Early Discharge of Patients with COVID-19 Symptoms in the Emergency Department.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (The Covid-19 pandemic) strains health care capacity. Better risk stratification, with discharge of patients with a predicted mild disease trajectory, can ease this burden. Elevated blood-soluble urokinase plasminogen activator receptor (suPAR) has previously been shown to be associated with risk of intubation in confirmed COVID-19 patients. OBJECTIVE: To evaluate whether point-of-care measures of suPAR in patients presenting to the emergency department (ED) with symptoms of COVID-19 can identify patients that can be safely discharged. METHODS: Observational cohort study including all patients in the ED with symptoms of COVID-19 from March 19 to April 3, 2020. SuPAR was measured at first presentation. Review of electronic patient records 14 days after admission was used to assess disease trajectory. Primary endpoints were mild, moderate, severe, or very severe trajectory. The predictive value of suPAR, National Early Warning Score (NEWS), C-reactive protein (CRP), and duration of symptoms was calculated using receiver operating characteristics (ROC). RESULTS: Of 386 patients, 171 (44%) had a mild disease trajectory, 79 (20%) a moderate, 63 (16%) a severe, and 73 (19%) a very severe disease trajectory. Low suPAR was a strong marker of mild disease trajectory. Results suggest a cut-off for discharge for suPAR < 2.0 ng/mL if suPAR is used as a single parameter, and <3.0 ng/mL when combined with NEWS ≤ 4 and CRP < 10 mg/L. CONCLUSION: suPAR is a potential biomarker for triage and safe early discharge of patients with COVID-19 symptoms in the ED. suPAR can be used even before SARS-CoV-2 status is known.

Longitudinal course of GDF15 levels before acute hospitalization and death in the general population.

Growth differentiation 15 (GDF15) is a potential novel biomarker of biological aging. To separate the effects of chronological age and birth cohort from biological age, longitudinal studies investigating the associations of GDF15 levels with adverse health outcomes are needed. We investigated changes in GDF15 levels over 10 years in an age-stratified sample of the general population and their relation to the risk of acute hospitalization and death. Serum levels of GDF15 were measured three times in 5-year intervals in 2176 participants aged 30, 40, 50, or 60 years from the Danish population-based DAN-MONICA cohort. We assessed the association of single and repeated GDF15 measurements with the risk of non-traumatic acute hospitalizations. We tested whether changes in GDF15 levels over 10 years differed according to the frequency of hospitalizations within 2 years or survival within 20 years, after the last GDF15 measurement. The change in GDF15 levels over time was dependent on age and sex. Higher GDF15 levels and a greater increase in GDF15 levels were associated with an increased risk of acute hospitalization in adjusted Cox regression analyses. Participants with more frequent admissions within 2 years, and those who died within 20 years, after the last GDF15 measurement already had elevated GDF15 levels at baseline and experienced greater increases in GDF15 levels during the study. The change in GDF15 levels was associated with changes in C-reactive protein and biomarkers of kidney, liver, and cardiac function. Monitoring of GDF15 starting in middle-aged could be valuable for the prediction of adverse health outcomes.

Association of GDF15 With Inflammation and Physical Function During Aging and Recovery After Acute Hospitalization: A Longitudinal Study of Older Patients and Age-Matched Controls.

Growth differentiation factor 15 (GDF15) is a stress-induced cytokine. Its plasma levels increase during aging and acute illness. In older Patients and age-matched Controls, we evaluated whether GDF15 levels (i) were associated with recovery after acute illness, and (ii) reflected different trajectories of aging and longitudinal changes in health measures. Fifty-two older Patients (≥65 years) were included upon admission to the emergency department (ED). At 30 days after discharge (time of matching), Patients were matched 1:1 on age and sex with Controls who had not been hospitalized within 2 years of inclusion. Both groups were followed up after 1 year. We assessed plasma levels of GDF15 and inflammatory biomarkers, frailty, nutritional status (mini nutritional assessment short-form), physical and cognitive function, and metabolic biomarkers. In Patients, elevated GDF15 levels at ED admission were associated with poorer resolution of inflammation (soluble urokinase plasminogen activator receptor [suPAR]), slowing of gait speed, and declining nutritional status between admission and 30-day follow-up. At time of matching, Patients were frailer and overall less healthy than age-matched Controls. GDF15 levels were significantly associated with participant group, on average Patients had almost 60% higher GDF15 than age-matched Controls, and this difference was partly mediated by reduced physical function. Increases in GDF15 levels between time of matching and 1-year follow-up were associated with increases in levels of interleukin-6 in Patients, and tumor necrosis factor-α and suPAR in age-matched Controls. In older adults, elevated GDF15 levels were associated with signs of accelerated aging and with poorer recovery after acute illness.

The effect of circadian-adjusted LED-based lighting on sleep, daytime sleepiness and biomarkers of inflammation in a randomized controlled cross-over trial by pragmatic design in elderly care home dwellers.

AIM: Elderly multimorbid care home dwellers are a heterogenic group of frail individuals that exhibit sleep disturbances and a range of co-morbidities. The project aimed to study the possible effect of indoor circadian-adjusted LED-lighting (CaLED) in the elderly residents' care home on their sleeping patterns and systemic biomarkers associated with inflammation. METHODS: A 16-week trial study was performed to follow the intervention and control groups using the Pittsburgh Sleep Quality Index (PSQI) and Epworth Sleepiness Scale (ESS) to monitor sleep and daytime sleepiness, and biomarkers IL-6, TNF-α and suPAR, to estimate the levels of inflammation. RESULTS: There was no significant impact on sleep improvement after the short intervention time when analyzing the PSQI and ESS results. However, we found several challenges using these tools for this specific group of individuals. Thus, important knowledge was gained for future studies in elderly care home dwellers. The inflammation state throughout the entire study period was stable for most of the elderly and no significant change was detected from before to after the intervention. This study represents a first-to-date attempt to ameliorate the adverse effects of sleep disturbances that characterize a randomly chosen group of elderly multimorbid subjects, by using circadian-adjusted LED-lighting in a natural care home environment. CONCLUSION: In this pragmatic randomized study of home dwelling individuals we were not able to demonstrate an improved sleep pattern as judged by PSQI, ESS or a change in inflammatory state.

Alterations of monocyte NF-κB p65/RelA signaling in a cohort of older medical patients, age-matched controls, and healthy young adults.

BACKGROUND: Altered monocyte NF-κB signaling is a possible cause of inflammaging and driver of aging, however, evidence from human aging studies is sparse. We assessed monocyte NF-κB signaling across different aging trajectories by comparing healthy older adults to older adults with a recent emergency department (ED) admission and to young adults. METHODS: We used data from: 52 older (≥65 years) Patients collected upon ED admission and at follow-up 30-days after discharge; 52 age- and sex-matched Older Controls without recent hospitalization; and 60 healthy Young Controls (20-35 years). Using flow cytometry, we assessed basal NF-κB phosphorylation (pNF-κB p65/RelA; Ser529) and induction of pNF-κB following stimulation with LPS or TNF-α in monocytes. We assessed frailty (FI-OutRef), physical and cognitive function, and plasma levels of IL-6, IL-18, TNF-α, and soluble urokinase plasminogen activator receptor. RESULTS: Patients at follow-up were frailer, had higher levels of inflammatory markers and decreased physical and cognitive function than Older Controls. Patients at follow-up had higher basal pNF-κB levels than Older Controls (median fluorescence intensity (MFI): 125, IQR: 105-153 vs. MFI: 80, IQR: 71-90, p < 0.0001), and reduced pNF-κB induction in response to LPS (mean pNF-κB MFI fold change calculated as the log10 ratio of LPS-stimulation to the PBS-control: 0.10, 95% CI: 0.08 to 0.12 vs. 0.13, 95% CI: 0.10 to 0.15, p = 0.05) and TNF-α stimulation (0.02, 95% CI: - 0.00 to 0.05 vs. 0.10, 95% CI: 0.08 to 0.12, p < 0.0001). Older Controls had higher levels of inflammatory markers than Young Controls, but basal pNF-κB MFI did not differ between Older and Young Controls (MFI: 81, IQR: 70-86; p = 0.72). Older Controls had reduced pNF-κB induction in response to LPS and TNF-α compared to Young Controls (LPS: 0.40, 95% CI: 0.35 to 0.44, p < 0.0001; and TNF-α: 0.33, 95% CI: 0.27 to 0.40, p < 0.0001). In Older Controls, basal pNF-κB MFI was associated with FI-OutRef (p = 0.02). CONCLUSIONS: Increased basal pNF-κB activity in monocytes could be involved in the processes of frailty and accelerated aging. Furthermore, we show that monocyte NF-κB activation upon stimulation was impaired in frail older adults, which could result in reduced immune responses and vaccine effectiveness.

Kinetics of the soluble urokinase plasminogen activator receptor (suPAR) in cirrhosis.

BACKGROUND: The soluble urokinase plasminogen activator receptor (suPAR) is related to hepatic inflammation and fibrosis and has been suggested to participate in the development of liver cirrhosis. Therefore, the aim of the current study was to measure the concentration of suPAR in the hepatic vein of cirrhotic patients during a liver vein catheterization to identify a possible hepatic suPAR generation. Furthermore, we explored if suPAR levels were associated with the degree of cirrhosis and liver dysfunction. METHODS AND PATIENTS: We included 105 cirrhotic patients and 19 liver-healthy controls. Blood was sampled from the hepatic vein and the femoral artery and suPAR was measured by enzyme-linked immunosorbent assay. RESULTS: We identified significantly higher median suPAR concentrations among the cirrhotic patients (7.2 ng/ml in the hepatic vein; 6.8 ng/ml in the femoral artery) compared to the controls (2.6 ng/ml, respectively, p-values <0.001). However, the median hepatic suPAR formation was 0.0 ng/ml in both groups. We observed significantly increasing suPAR levels according to higher Child classes (4.5 ng/ml, 6.9 ng/ml and 9.0 ng/ml, Child A, B, C respectively; p-value<0.001), and significantly higher median suPAR concentrations in patients with ascites versus patients without ascites (8.1 ng/ml versus 5.3 ng/ml, respectively, p-value<0.001). suPAR levels were significantly related to bilirubin (r = 0.48, p<0.001), the hepatic venous pressure gradient (r = 0.39, p<0.001), the cardiac index (r = 0.24, p = 0.02) and the plasma volume (r = 0.33, p = 0.001), whereas suPAR levels were significantly inversely related to albumin (r = -0.59, p<0.001), plasma coagulation factors (r-0.39, p<0.001), the mean arterial pressure (r = -0.28, p = 0.004), the systemic vascular resistance (r = 0.26, p = 0.007), the indocyanine green clearance (r = -0.51, p<0,001) and the galactose elimination capacity (r = -0.39, p<0.001). CONCLUSION: We identified elevated suPAR concentration in cirrhotic patients, which correlated significantly with the degree of cirrhosis and liver failure, but we were not able to demonstrate hepatic suPAR generation per se. This suggests that further investigations of the source of suPAR in cirrhotic patients need to be undertaken.

Aging and lineage allocation changes of bone marrow skeletal (stromal) stem cells.

Aging is associated with decreased bone mass and accumulation of bone marrow adipocytes. Both bone forming osteoblastic cells and bone marrow adipocytes are derived from a stem cell population within the bone marrow stroma called bone marrow stromal (skeletal or mesenchymal) stem cells (BMSC). In the present review, we provide an overview, based on the current literature, regarding the physiological aging processes that cause changes in BMSC lineage allocation, enhancement of adipocyte and defective osteoblast differentiation, leading to gradual exhaustion of stem cell regenerative potential and defects in bone tissue homeostasis and metabolism. We discuss strategies to preserve the "youthful" state of BMSC, to reduce bone marrow age-associated adiposity, and to counteract the overall negative effects of aging on bone tissues with the aim of decreasing bone fragility and risk of fractures.

Low Constitutive Cell Surface Expression of HLA-B Is Caused by a Posttranslational Mechanism Involving Glu180 and Arg239.

HLA class I cell surface expression is crucial for normal immune responses, and variability in HLA expression may influence the course of infections. We have previously shown that classical HLA class I expression on many human cell types is biased with greatly reduced expression of HLA-B compared with HLA-A in the absence of inflammatory signals. In the search for the mechanisms responsible for this discrepancy, we have recently reported that the regulation is mainly posttranslational and that the C-terminal part of the α2 domain and the α3 domain contain the molecular determinants that explain most of the variability of expression between common HLA-A and -B allomorphs. In this study, we present a fine mapping of the structural determinants that allow such variability by exchanging key amino acids located within the C-terminal part of the α2 domain and the α3 domain of HLA-A2 and -B8, including Glu/Asp at position 177, Gln/Glu at position 180, Gly/Arg at position 239, and Pro/Ser at position 280. We found that the HLA-A2 and -B8 expression profiles could be interconverted to a large extent by mutual exchange of Gln/Glu at position 180 or by Gly/Arg at position 239. The presence of Gln180 and Gly239, as in HLA-A2, led to higher cell surface expression levels when compared with the presence of Glu180 and Arg239, as in HLA-B8. This indicates that the amino acids at positions 180 and 239 determine the level of cell surface expression of common HLA-A and -B allomorphs, probably by affecting HLA processing in the Ag presentation pathway.

Cell surface expression level variation between two common Human Leukocyte Antigen alleles, HLA-A2 and HLA-B8, is dependent on the structure of the C terminal part of the alpha 2 and the alpha 3 domains.

Constitutive cell surface expression of Human Leukocyte Antigen (HLA) class I antigens vary extremely from tissue to tissue and individual antigens may differ widely in expression levels. Down-regulation of class I expression is a known immune evasive mechanism used by cancer cells and viruses. Moreover, recent observations suggest that even minor differences in expression levels may influence the course of viral infections and the frequency of complications to stem cell transplantation. We have shown that some human multipotent stem cells have high expression of HLA-A while HLA-B is only weakly expressed, and demonstrate here that this is also the case for the human embryonic kidney cell line HEK293T. Using quantitative flow cytometry and quantitative polymerase chain reaction we found expression levels of endogenous HLA-A3 (median 71,204 molecules per cell) 9.2-fold higher than the expression of-B7 (P = 0.002). Transfection experiments with full-length HLA-A2 and -B8 encoding plasmids confirmed this (54,031 molecules per cell vs. 2,466, respectively, P = 0.001) independently of transcript levels suggesting a post-transcriptional regulation. Using chimeric constructs we found that the cytoplasmic tail and the transmembrane region had no impact on the differential cell surface expression. In contrast, ~65% of the difference could be mapped to the six C-terminal amino acids of the alpha 2 domain and the alpha 3 domain (amino acids 176-284), i.e. amino acids not previously shown to be of importance for differential expression levels of HLA class I molecules. We suggest that the differential cell surface expression of two common HLA-A and-B alleles is regulated by a post-translational mechanism that may involve hitherto unrecognized molecules.

Separate developmental programs for HLA-A and -B cell surface expression during differentiation from embryonic stem cells to lymphocytes, adipocytes and osteoblasts.

A major problem of allogeneic stem cell therapy is immunologically mediated graft rejection. HLA class I A, B, and Cw antigens are crucial factors, but little is known of their respective expression on stem cells and their progenies. We have recently shown that locus-specific expression (HLA-A, but not -B) is seen on some multipotent stem cells, and this raises the question how this is in other stem cells and how it changes during differentiation. In this study, we have used flow cytometry to investigate the cell surface expression of HLA-A and -B on human embryonic stem cells (hESC), human hematopoietic stem cells (hHSC), human mesenchymal stem cells (hMSC) and their fully-differentiated progenies such as lymphocytes, adipocytes and osteoblasts. hESC showed extremely low levels of HLA-A and no -B. In contrast, multipotent hMSC and hHSC generally expressed higher levels of HLA-A and clearly HLA-B though at lower levels. IFNγ induced HLA-A to very high levels on both hESC and hMSC and HLA-B on hMSC. Even on hESC, a low expression of HLA-B was achieved. Differentiation of hMSC to osteoblasts downregulated HLA-A expression (P = 0.017). Interestingly HLA class I on T lymphocytes differed between different compartments. Mature bone marrow CD4(+) and CD8(+) T cells expressed similar HLA-A and -B levels as hHSC, while in the peripheral blood they expressed significantly more HLA-B7 (P = 0.0007 and P = 0.004 for CD4(+) and CD8(+) T cells, respectively). Thus different HLA loci are differentially regulated during differentiation of stem cells.

Impaired TCA cycle flux in mitochondria in skeletal muscle from type 2 diabetic subjects: marker or maker of the diabetic phenotype?

The diabetic phenotype is complex, requiring elucidation of key initiating defects. Recent research has shown that diabetic myotubes express a primary reduced tricarboxylic acid (TCA) cycle flux. A reduced TCA cycle flux has also been shown both in insulin resistant offspring of T2D patients and exercising T2D patients in vivo. This review will discuss the latest advances in the understanding of the molecular mechanisms regulating the TCA cycle with focus on possible underlying mechanism which could explain the impaired TCA flux in insulin resistant human skeletal muscle in type 2 diabetes. A reduced TCA is both a marker and a maker of the diabetic phenotype.

Decellularized matrix from tumorigenic human mesenchymal stem cells promotes neovascularization with galectin-1 dependent endothelial interaction.

BACKGROUND: Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC) strain (hMSC-TERT20) immortalized by retroviral vector mediated human telomerase (hTERT) gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+) and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. CONCLUSIONS: Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1 expression was required to bring about matrix-endothelial interactions and for xenografted hMSC -BD11 cells to optimally recruit host vasculature.

Human myotubes from myoblast cultures undergoing senescence exhibit defects in glucose and lipid metabolism.

Adult stem cells are known to have a finite replication potential. Muscle biopsy-derived human satellite cells (SCs) were grown at different passages and differentiated to human myotubes in culture to analyze the functional state of various carbohydrate and lipid metabolic pathways. As the proliferative potential of myoblasts decreased dramatically with passage number, a number of cellular functions were altered: the capacity of myoblasts to fuse and differentiate into myotubes was reduced, and metabolic processes in myotubes such as glucose uptake, glycogen synthesis, glucose oxidation and fatty acid ß-oxidation became gradually impaired. Upon insulin stimulation, glucose uptake and glycogen synthesis increased but as the cellular proliferative capacity became gradually exhausted, the response dropped concomitantly. Palmitic acid incorporation into lipids in myotubes decreased with passage number and could be explained by reduced incorporation into diacyl- and triacylglycerols. The levels of long-chain acyl-CoA esters decreased with increased passage number. Late-passage, non-proliferating, myoblast cultures showed strong senescence-associated ß-galactosidase activity indicating that the observed metabolic defects accompany the induction of a senescent state. The main function of SCs is regeneration and skeletal muscle-build up. Thus, the metabolic defects observed during aging of SC-derived myotubes could have a role in sarcopenia, the gradual age-related loss of muscle mass and strength.